Hybridization probes used for nucleic acid detection generally are single-stranded molecules complementary to a nucleic acid sequence sought to be detected (“target sequence”). Background descriptions of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. No. 5,840,488. Hybridization probes may be labeled with detectable moieties such as radioisotopes, antigens or chemiluminescent moieties. When a first single strand of nucleic acid contains sufficient contiguous complementary bases to a second, and the two strands are brought together under conditions which promote hybridization, double stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed.
Molecular beacons are examples of hybridization probes that have limited regions of self-complementarity. These probes, which are particularly useful for conducting homogeneous detection assays, comprise a target-complementary “loop” portion, a “stem” portion formed by the annealing of two complementary “arms” that extend from the loop, a fluorophore group and a quencher group. The fluorophore is typically linked to the end of one arm while the quencher is typically linked to the end of the other arm. The stem portion maintains the probe in a closed conformation in the absence of a target nucleic acid sequence, so that energy received by the fluorophore is transferred to the quencher, rather than being emitted. Upon hybridizing a target polynucleotide, the complementary arm sequences of the molecular beacon become separated, thereby shifting the probe to an open conformation. This shift is detectable as a flourescent signal resulting from the reduced energy transfer between the fluorophore and the quencher (see Tyagi et al., Nature Biotechnology 14:303 (1996); Fang et al., Analytical Chemistry, Dec. 1, 2000 issue:747A). Molecular beacons are fully described in U.S. Pat. Nos. 5,925,517 and 6,150,097, the disclosures of which are hereby incorporated by reference.
Molecular beacons are not limited to having conventional nucleic acid constituents. In addition to standard nucleotides, peptide nucleic acids (PNAs) have also been used for preparing molecular beacons (see published International Patent Application No. PCT/US98/22785). Regardless of whether conventional nucleotides or PNA analogs were used to prepare these probes, stem regions uniformly were complementary as the result of antiparallel pairing of nucleobases disposed on sugar-phosphate or glycyl peptide backbones.
Molecular beacon probe design is naturally rendered somewhat more complicated than the process of designing linear probes due to the added presence of the stem structure. Since the stem portions of previously described molecular beacons comprised base moieties that conceivably could interact through complementary pairing with bases present in the target polynucleotide that is to be detected, those interactions must be considered during the design of every molecular beacon. Thus, the process of designing a molecular beacon requires selection of a target-complementary sequence for the loop portion of the probe, as well as consideration of the effect that the base sequence of the stem portion will have on interaction with the target polynucleotide that is to be detected.
Previous attempts to simplify the process of designing molecular beacons have focused on the use of a “universal stem” structure. For example, in U.S. Pat. No. 6,103,476, Tyagi et al., described stems consisting of arm regions that comprised nucleobase sequences orientated by standard antiparallel complementarity, with one of the arms being linked to a fluorophore and the other arm being linked to a quencher moiety. In these constructs it remained possible for nucleobases of the universal stem to influence hybridization between the target polynucleotide and the molecular beacon probe, for example by influencing the Tm of the probe:target complex. Notably, this same feature would also characterize universal stems comprised of PNAs because the nucleobases of the denatured stem could still interact with the target sequence.
The present invention provides a new class of hybridization probes wherein opportunities for complementary interactions between nucleobases of a target polynucleotide and nucleobases of the stem region of a molecular beacon are substantially eliminated. Additionally, these new probes have been shown to have unique properties that distinguish them from previously known hybridization probes.